Live Chat Support Software
800.227.0627
PEGylation
PEGylation

PEGylation for enhancing drug delivery

Bio-Synthesis offers synthetic polymer PEG (polyethylene glycol) modification and bioconjugation services by site specific ligation of polyethylene glycol to peptide/protein, oligonucleotide, antibodies, lectins, enzymes, toxins, drugs or other small molecules as delivery technology in variety of applications. The conjugation of PEG to a peptide or protein has shown the possibility of improving the pharmacokinetic profile of a linked drug. In the last few years this technology, firstly developed for proteins, has been transferred to non-peptide drugs, opening a new area of investigation that is now receiving increasing interest. This leads to new opportunities for many therapeutic treatments as it is possible to use molecules that could not before be exploited due to limitations such as inadequate water solubility, high nonspecific toxicity and poor pharmacokinetic profiles.

Bio-Synthesis provides reliable and flexible chemical biology solutions that cover almost all aspects you may encounter during the preparation phase in diagnostic and drug discovery research. Our unique approach for a homogeneous PEGylated populations where the specific PEGylation site can be selected and controlled, thus improving efficacy compared to existing PEGylation techniques. All synthetic bioconjugation projects are meticulously monitored according to Bio-Synthesis's stringent quality assurance and quality control standard that are fully backed up by an bioanalytical laboratory.

What is PEG (Polyethylene glycol) Polymer

PEG(polyethylene glycol, the most often used polymer, is a chemical compound composed of repeating ethylene glycol units. It also known as poly ethylene oxide (PEO) and polyoxyethylene (POE) with molecular weights from 300g/mol to 10,000,000 g/mol. with general structure HO-(CH2CH2O)n-H.

Conjugation of synthetic PEG with a biopolymer is sometimes called "bio-hybrid" or "macromolecular chimera. Covalent modification of a PEG polymer to a therapeutic molecule is called PEGylation. As peptide and protein based therapeutics have become a continual focus for innovative drug discovery, they offer key advantages over small molecules (e.g. lower toxicity profiles, and increased specificity and affinity to target). There are, however, a few setbacks in the development of effective peptide drugs including short half-life, rapid glomerular filtration, and degradation by proteolytic enzymes. PEGylation is the reaction between the functional group of activated polyethylene glycol and reactive residues on the protein surface (such as lysine). It is a popular avenue used to overcome the disadvantages associated with protein based drugs. PEGylation increases the therapeutic value of peptides and proteins—PEG acts as a shield to protect the biopolymers from proteolysis, increases the overall molecular weight which increases circulation half-life, decreases immunogenicity by limiting uptake into dendritic cells (PEG itself is non-immunogenic) and increases solubility of the protein. PEG polymers are available in various sizes for the tuning of PEG-drug conjugate circulating half-lives and solubility.

Limitations:

Traditional PEGylation involves the reaction of activated PEG with a free amine such as lysine on the peptide or protein surface or at the N-terminus. This non-site selective route leads to multiple attachments because peptides and proteins can have several free amines. As a result, the conjugated product can contain a mixture of molecules with PEG linked to various free amines. The homogeneity of PEG conjugated products is compromised, making it difficult to obtain reproducible monosubstituted PEG conjugates with the same effective biological activities to fulfill regulatory criteria. An important consideration in PEGylation is the relationship between the reaction site and bioactivity, i.e. receptor binding may take place at the N-terminus or at other sites that are also reactive with activated PEG. Non-site specific PEGylation may also lead to a product in which the conjugated PEG masks or interferes with the receptor binding site, decreasing bioactivity up to 100 fold in comparison to the unmodified protein. Thus, it is important to consider controllable site-specific routes that offer high product yield and reproducibility while preserving bioactivity.

Site-Specific PEGylation

In recent times, site-specific PEGylation is used to overcome the limitations of non-selective strategies. A number of approaches for site-specific PEGylation have been developed to allow for the incorporation of a single PEG moiety at a defined position within the protein sequence. Site-specific PEGylation minimizes interference of biological function and affords homogenous PEGylated products that are more easily purified and reproduced. The general approach to site-specific PEGylation involves the modification or addition of an amino acid that reacts exclusively with the PEG. A common example is the addition of a cysteine to introduce a free reactive thiol. Other chemical routes include the blocking of reactive residues, N or C-terminal modification (e.g. introduction of a His tag, reductive alkylation, introduction of a reactive hydrazide), click chemistry, and Staudinger ligation to name a few. The chemistry is determined based on the protein or peptide sequence. Our chemists at Bio-Synthesis are ready to work with you on your PEGylation project scope and design.

Contact our Technical Service Center at 800.220.0627 or contact us online with your detail project specifications, a project manager will be assigned to help you with design and develop an appropriate synthetic method for your specific needs.

Custom PEGylation Portfolios

  • Peptide PEGylation
  • Peptide SMA conjugation
  • SMA protein conjugation
  • PEGylation with PEG to antibodies
  • PEG conjugated oligonucleotide
  • Peptide dextran bioconjugation
  • Protein modification with activated dextrans
  • Coupling polyaldehyde dextran to proteins
  • PEG-O-Drug
  • Drug-O-PEG-O-Drug
  • poly(styrene-co-maleic acid/anhydride) (SMA)-drug conjugates
  • N-(2-hydroxypropyl) methacrylamide (HPMA) Peptide conjugation

Sample Submission Requirement:

Biomolecule supplied by customers should be sufficiently pure. Please provide 5 mgs of starting material with the necessary data for purity assessment. Commercially available biopolymers can be supplied by customers or synthesize or ordered through Bio-Synthesis.

Price

Price varies based on the project specifications. Our service includes materials and labor for conjugation only! Price does not include the cost of biopolymer synthesis or order through Bio-Synthesis from an commercial vendors and, if deemed necessary, biopolymer modification to introduce additional functional groups, extra linkers, spacers. Please contact us for a quote.

PEGylation Service Descriptions

Chemistry:

Coupling of preactivated small molecule and biomolecule with chemical reactive groups such as amine, thiol, carboxylate, hydroxyl, aldehyde and ketone, active hydrogen through use of various cross linkers.

Type of PEG for Conjugation

  • Linear Activated PEGs
  • Multi-arm PEG conjugation
  • Branched PEG conjugation
  • Heterofunctional PEGs conjugation
  • Comb-Shaped Copolymers
  • PEG containing Biotin

Service Specification:

After standard desalting, or purification, a small percent of heterogeneous products containing single or multi-site conjugate per molecule may exist.

Procedure:

After labeling, final conjugates must first be isolated from excess or unreacted reagent by gel filtration or dialysis. In many cases, simple dialysis may suffice to remove unreacted reagent from the reaction solution. Additional purification technique such as stirred cell filtration, tangential flow filtration (TFF), gel filtration chromatography may also be used to either remove excess reagent or isolate and characterized the cross-linked product. For reagents (mostly protein and other biological molecules) that are similar in size or larger than the antibody, one must resort to other purification techniques such as affinity chromatography, ion-exchange chromatography, and hydrophobic interaction chromatography.

Cross-linked target molecules may then be further characterized by gel electrophoresis. It may be subject to additional analyses with an additional fee. This including spectroscopic (MALDI-TOF, ESI, LC-MS Fluorescence), electrophoresis, immunochemical biochemical, enzymatical analysis and TLC. QC (quality control) and QA (quality assurance) procedures are also followed independently to offer you double guarantee for the highest quality possible of every delivered conjugates. Moreover, our dedicated technical account managers will guide your project through every step of the process and constantly keep you informed of the latest project progress.

Ordering and Submitting Requests for Bioconjugation Services

For us to better understand your customized project, please complete our Bioconjugation Service Questionnaire. The more our chemists understand your project needs, the more accurate feedback we will be able to provide you.  Provide us with your project details will enable to us to recommend the best reagents to use for your project.  The most useful and readily available tools for bioconjugation projects are cross-linking reagents. A large number of cross-linkers, also known as bifunctional reagents, have been developed.  There are several ways to classify the cross-linkers, such as the type of reactive group, hydrophobicity or hydrophilicity, and the length of the spacer between reactive groups.  Other factors to consider are whether the two reactive groups are the same or different (for example, heterobifunctional or homobifunctional reagents), whether the spacer is cleavable, and whether the reagents are membrane permeable or impermeable.  The most accessible and abundant reactive groups in proteins are the ϵ-amino groups of lysine.  Therefore, a large number of the most common cross-linkers are amino selective reagents, such as imidoesters, sulfo-N-hydroxysuccinimide esters, and N-hydroxysuccinimide esters.  Due to the high reactivity of the thiol group with N-ethylmaleimide, iodoacetate and a-halocarbonyl compounds, new cross-linkers have been developed that contain maleimide and a-carbonyl moieties.  Usually, N-alkylmaleimides aremore stable than their N-aryl counterparts.

In addition to the reactive groups on the cross-linkers, a wide variety of connectors and spacer arms have also been developed.  The nature and length of the spacer arm play an important role in the functionality.  Longer spacer arms are generally more effective when coupling large proteins or those with sterically protected reactive side-chains.  Other important considerations are the hydrophobicity, hydrophilicity, and the conformational flexibility.  Long aliphatic chains generally fold on themselves when in an aqueous environment, which makes the actual distance spanned by such linker arms less than expected.  Instead, spacers that contain more rigid structures (for example, aromatic groups or cycloalkanes) should be used.  These structures, however, tend to be very hydrophobic which could significantly decrease the solubility of the modified molecules or even modify some of their properties.  In such cases, it is recommended to choose a spacer that contains an alkylether (PEO) chain.  Bio-Synthesis offers several cross-linkers with PEO chains, such as thiol-binding homobifunctional reagents, heterobifunctional based, and their derivatives.

Within 3-5 days upon receiving your project scope, we will provide you an appropriate quotation. An order can be placed with PO (Purchase Order) or major credit cards ( ). Your credit card will be billed under Bio-Synthesis, Inc.